Dual endogenous enzyme block solution

Immunohistochemical (IHC) analysis of tumor xenograft samples was followed a previously published protocol (12 (link)) using primary antibodies against TBX2, WNT3A, MMP9, and FOXA1. Briefly, FFPE sections (4μm) were deparaffinized, rehydrated, and subjection to antigen retrieval. Following incubation with Dual Endogenous Enzyme Block solution (Dako) for 10 minutes, the section was treated with primary antibody of varying dilutions using Antibody Diluent solution (Dako) at 4° C overnight. The section was then washed 3 times with PBST (PBS containing 0.2% Tween-20) for 5 minutes per wash. The section was then treated with EnVision + Dual Link System-HRP (Dako) for 30 min to detect specific staining. The sections were then washed 3 times for 5 minutes each, and developed with 3′3-diaminobenzidine (Dako). Image acquisition was performed using a Nikon camera and software. Magnification: x500. IHC staining intensity was scored using the combined intensity and percentage of positive-scoring cells as previously reported (13 (link)). Strong intensity was scored as 3, intermediate as 2, weak as 1, and negative as 0 Each intensity score was then summed with the percentage of cells that were stained, with >50% of the cells as 2, <50% of the cells as 1, and none as 0.